My goal is to determine how "pioneer" or initial chromatin binding transcription factors engage target sites in silent, compacted chromatin and promote entry of subsequent proteins. The pioneer transcription factor HNF3 binds to and activates numerous liver genes required for differentiation and metabolism. Previous studies have shown that HNF3 engages specific sites in chromatin in undifferentiated endoderm, prior to the activation of the albumin gene during liver specification. Using an in vitro system of reconstituted nucleosomes, I have demonstrated that HNF3 can bind its target sites within highly compacted chromatin packaging the albumin enhancer. This binding, and subsequent nucleosome positioning, creates a localized, open domain of chromatin accessible to other proteins. I hypothesize that chromatin binding and remodeling initiated by HNF3 establishes a chromatin environment that enables the subsequent binding, remodeling, and modification events required for albumin enhancer activation. I also propose that establishment of HNF3-mediated transcriptional competency is a prerequisite to the developmental activation of genes required for hepatocyte differentiation and function. In order to test these hypotheses, I will combine in vitro biochemical and in vivo-genetic approaches to address the following specific aims: 1) Does HNF3 displace linker histone from the albumin enhancer during chromatin opening? 2) Can HNF3-mediated chromatin remodeling activate the albumin enhancer in vivo? and 3) Is HNF3-mediated transcriptional competency essential for the activation of liver genes? These studies are designed to define the role played by pioneer DNA binding regulatory factors in the chromatin remodeling required for gene activation. Understanding how chromatin structural changes specify early gene regulatory events will provide insight into molecular mechanisms which underlie growth, development, and disease in humans. This primary goal of this grant is to determine the mechanisms used by the HNF3 protein to activate genes in the liver. HNF3 is essential for liver development, and is mutated in certain forms of cancer and diabetes. The results of the studies described in this grant will provide novel targets for the study and treatment of liver disease and neoplasia.